The goal of this proposal is to determine the mechanisms by which genes can be efficiently transferred into hematopoietic stem cells (HSC) without concurrent loss of stem cell function. Based on the results from human clinical gene therapy trials, the level of retroviral transduction of HSC- ie pluripotent progenitors able to contribute to long term hematopoiesis- is currently inadequate for therapeutic benefit. The low efficiency of transduction using Moloney Murine Leukemia Virus (MoMuLV) based vectors is assumed to be secondary to non- integration of provirus into the genomes of quiescent HSC. An additional postulated limitation to transduction is low expression of viral receptors on HSC. The great majority of hematopoietic progenitors are cytokine responsive, efficiently transduced during short exposure to virus and do not contribute to long term multilineage hematopoiesis. Standard in vitro assays measure these mature progenitors. We have determined that the CD34+CD38-immunophenotype of bone marrow and cord blood, although used to enrich for HSC, defines a functionally heterogeneous population in terms of cytokine responsiveness [using the Extended Long Term Culture-Initiating Cell (ELTC-IC) assay], lineage potential (using a single cell assay of pluripotentiality ie B lymphoid and myeloid potential) and the ability to be transduced. We hypothesize that by studying the rare, functionally primitive and transduction resistant HSC within the heterogeneous CD34+CD38-progenitor pool, we may determine the mechanisms by which this population escape transduction and develop the means by which gene transfer can be improved. Three approaches to increase HSC transduction will be evaluated: prolonging exposure to virus, manipulation of the HSC in vitro, and manipulation of the virus and its envelope. The Specific Aims of this proposal are as follows: (1) To determine the period in vitro during which HSC stem cell function (pluripotentiality) is maintained, (2) To determine the effects of manipulating invitro conditions on the efficiency of retroviral transduction, expression of viral receptors and the function of HSC, and (3) To determine the efficiency of transduction of HSC using a lentiviral vector pseudotyped with a Vesicular Stomatitis Virus (VSV) envelope. These studies will reveal the mechanisms for resistance of HSC to retroviral transduction and provide novel means for improvement of transduction efficiency.